Cellular Lysis and Protein Analysis

The cells were harvested and washed with ice-cold PBS (phosphate buffered saline, Caisson Labs), followed by lysis with Bullet Blender® (Next Advance, Inc.) in PLC lysis buffer [13 (link), 35 (link)]: 30 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EGTA, 10 mM NaPPi, 100 mM NaF, 1 mM Na₃VO₄ supplemented with protease inhibitor cocktail (Roche), 1 mM PMSF, 10 μM TSA (Trichostatin A, Selleckchem), and 5 mM nicotinamide (Alfa Aesar). Total protein concentrations of the cell lysates were determined using the DC protein assay (Bio-Rad). Western blot and image analysis were conducted as described previously [13 (link)]. Antibody catalog numbers and vendors are as follows: C1 (A31857) and C3 (A21362) from Invitrogen; BNIP3 (ab10433) from Abcam; β-actin (MA5-15739), GAPDH (MA5-15738), and LC3 (PA1-16931) from Pierce; Mfn1 (sc-50330), Mfn2 (sc-50331), and Tfam (sc-28200) from Santa Cruz; PGC1α (Ab3242) from Millipore; VDAC (4661s) and Drp1 (8570) from Cell Signaling Technology; Beclin-1 3738s and Beclin-1 MABN16 from Cell Signaling Technology and Millipore, respectively; NRF1 (LS-B43) from LifeSpan BioSciences.